1. Field of the Invention
This invention relates to the area of genetic diagnosis. More specifically, the invention relates to detection of an alteration of wild-type mitochondrial ribosomal RNA(rRNA) which is associated with ototoxic deafness in humans.
2. Description of Related Art
Sensorineural deafness, either in conjunction with neuromuscular diseases or with diabetes, has been associated with heteroplasmic mitochondrial DNA (mtDNA) mutations (Shoffner, et al., Adv. Human Genet., 19:267-330, 1990; Ballinger, et al., Nature Genet., 1:11-15, 1992; van den Ouweland, et al., Nature Genet., 1:368-371, 1992; Reardon, et al., Lancet, 34:1376-1379, 1992). The likelihood of mtDNA mutations has also been suggested in two forms of non-syndromic deafness. A maternal inheritance pattern has been reported in several pedigrees in the Far East with familial aminoglycoside-induced deafness (Higashi, Clin. Genet., 35:433-436, 1989; Hu, et al., J. Med. Genet., 28:79-83, 1991), and a single large Arab-Israeli pedigree with maternally-inherited congenital deafness has been described (Jaber, et al., J. Med. Genet., 29:86-90, 1992).
Mitochondrial DNA is transmitted exclusively through mothers, since the sperm apparently contributes no mitochondria to the zygote. This leads to the expectation that a defect in a mitochondrial gene should lead to disease equally in both sexes, but can only be transmitted through the maternal line. There are hundreds of mitochondria in each cell and they serve a variety of metabolic functions, the most important being the synthesis of ATP by oxidative phosphorylation. Each mitochondrion contains several mitochondrial DNA (mtDNA) chromosomes, which in humans are 16,569 basepairs (bp) double-stranded circles. Replication, transcription, and translation of the mtDNA occurs within the mitochondrion. The mitochondrial DNA encodes 13 messenger RNAs, and the large and small ribosomal RNAs and 22 transfer RNAs which are necessary for their translation. The messenger RNAs are translated on mitochondrion-specific ribosomes, using a mitochondrion-specific genetic code, into 13 proteins. These proteins interact with approximately sixty nuclear encoded proteins to form the five enzyme complexes required for oxidative phosphorylation.
Irreversible hearing loss is the main complication of aminoglycoside antibiotics such as streptomycin, gentamicin, and kanamycin (Sande, et al., Antimicrobial agents, in: Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition. eds, Gilman, et al., Pergamon Press, Inc., Elmsford, N.Y., pp 1098-1116, 1990). In China, due to widespread use of aminoglycosides, nearly 25% of all deaf-mutes in one district of Shanghai could trace the cause of hearing loss to aminoglycoside usage (Hu, et al., J. Med. Genet., 28:79-93, 1991). Of these patients, roughly 1/4 had other relatives with ototoxic deafness. In all of the 22 cases in which vertical transmission of this susceptibility could be traced, the inheritance pattern matched that of a mitochondrially-inherited trait, i.e., being transmitted only through females. A similar situation occurred in Japan, where Higashi tabulated 28 families with streptomycin-induced deafness, and found that in all but two cases, the susceptibility trait was maternally-inherited (Higashi, Clin. Genet., 35:433-436, 1989). Hu, et al., supra also noted that the majority of familial cases received antibiotics for a much shorter period than the sporadic cases, implying the presence of a predisposing mutation or genetic susceptibility (Hu, et al., supra).
The mitochondrial ribosome in the cochlea is the most likely target of aminoglycoside ototoxicity, since the "natural target" of aminoglycosides is the evolutionarily-related bacterial ribosome (Sande, et al., supra). In bacterial studies, aminoglycosides appear to stabilize mismatched aminoacyl-tRNAs in the 70S ribosome, allowing misreading of the mRNA during translation (Hornig, et al., Biochimie, 69:803-813, 1987). In addition to their interactions with ribosomal proteins, aminoglycosides bind to the E. coli 16S rRNA, as demonstrated by chemical protection and crosslinking experiments (Moazed, et al., Nature, 327:389-394, 1987; Gravel, et al., Biochemistry, 26:6227-6232, 1987). These physical experiments predict regions of the small rRNA which are important in translational fidelity. Their relevance has been borne out by the isolation of aminoglycoside-resistance mutations in bacteria, yeast mitochondria, Tetrahymena, and chloroplasts which map to the predicted regions of the evolutionarily conserved small rRNA (Tzagaloff, et al., J. Biol. Chem., 257:5921-5928, 1982; Spangler, et al., J. Biol. Chem., 260: 6334-6340, 1985; Gauthier, et al., Mol. Gen. Genet., 214:192-197, 1988; Melancon, et al., Nucl. Acids Res., 16:9631-9639, 1988). Thus, the mitochondrial rRNA genes, and especially the corresponding 12S rRNA gene, become prime candidates for the site of the mtDNA mutation in maternally-inherited aminoglycoside-induced deafness.
Although the role of the mitochondrial DNA in ototoxic deafness has been suspected, no adequate diagnostic test was available to identify those individuals at risk. Consequently, at least where the ototoxin is an aminoglycoside antibiotic, physicians have been faced with the dilemma of choosing between the use of the antibiotic and the potential irreversible loss of hearing which might occur in the patient. The present invention addresses this problem by providing a rapid, non-invasive, and highly accurate diagnostic test which can identify individuals at risk for ototoxic deafness.